The number of mononuclear cells isolated from spleen samples was standardized to 5 x 106 and used for RNA isolation with the use of the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. Genomic DNA remaining in the samples after RNA isolation was digested with deoxyribonuclease I (Sigma Aldrich, USA). The concentration of eluted RNA was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and the samples were stored at -80°C until further analyzed.
Reverse transcription was carried out with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, USA) according to the manufacturer’s recommendations. The concentration of RNA was standardized to 0.5 μg per sample. The relative expression of the genes encoding CD4 and CD8 T cells receptors and IFN-γ was determined by qPCR method described previously [21 (link)] with the use of Power SYBR Green PCR Master Mix kit (Life Technologies, USA) and LightCycler 96 (Roche, Switzerland).
Reverse transcription was carried out with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, USA) according to the manufacturer’s recommendations. The concentration of RNA was standardized to 0.5 μg per sample. The relative expression of the genes encoding CD4 and CD8 T cells receptors and IFN-γ was determined by qPCR method described previously [21 (link)] with the use of Power SYBR Green PCR Master Mix kit (Life Technologies, USA) and LightCycler 96 (Roche, Switzerland).
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